bioRxiv2025
KRAS is a pivotal oncoprotein that regulates cell proliferation and survival through interactions with downstream effectors such as RAF1. Oncogenic mutations in KRAS, including G12V, G13D, and Q61R, drive constitutive activation and hyperactivation of signaling pathways, contributing to cancer progression. Despite significant advances in understanding KRAS biology, the structural and dynamic mechanisms of KRAS binding and allostery by which oncogenic mutations enhance KRAS-RAF1 binding and signaling remain incompletely understood. In this study, we employ microsecond molecular dynamics simulations, Markov State Modeling, mutational scanning and binding free energy calculations together with dynamic network modeling to elucidate the effect of KRAS mutations and characterize the thermodynamic and allosteric drivers and hotspots of KRAS binding and oncogenic activation. Our simulations revealed that oncogenic mutations stabilize the open active conformation of KRAS by differentially modulating the flexibility of the switch I and switch II regions, thereby enhancing RAF1 binding affinity. The G12V mutation rigidifies both switch I and switch II, locking KRAS in a stable, active state. In contrast, the G13D mutation moderately reduces switch I flexibility while increasing switch II dynamics, restoring a balance between stability and flexibility. The Q61R mutation induces a more complex conformational landscape, characterized by the increased switch II flexibility and expansion of functional macrostates, which promotes prolonged RAF1 binding and signaling. Mutational scanning of KRAS-RAF1 complexes identified key binding affinity hotspots, including Y40, E37, D38, and D33, and together with the MM-GBSA analysis revealed the hotspots leverage synergistic electrostatic and hydrophobic binding interactions in stabilizing the KRAS-RAF1 complexes. Network-based analysis of allosteric communication identifies critical KRAS residues (e.g., L6, E37, D57, R97) that mediate long-range interactions between the KRAS core and the RAF1 binding interface. The central {beta}-sheet of KRAS emerges as a hub for transmitting conformational changes, linking distant functional sites and facilitating allosteric regulation. Strikingly, the predicted allosteric hotspots align with experimentally identified allosteric binding hotspots that define the energy landscape of KRAS allostery. This study highlights the power of integrating computational modeling with experimental data to unravel the complex dynamics of KRAS and its mutants. The identification of binding hotspots and allosteric communication routes offers new opportunities for developing targeted therapies to disrupt KRAS-RAF1 interactions and inhibit oncogenic signaling. Our results underscore the potential of computational approaches to guide the design of allosteric inhibitors and mutant-specific therapies for KRAS-driven cancers.